No treatment or no satisfactory treatment exists for numerous lethal and/or highly debilitating diseases associated with disregulated activity of NF-κB molecules, including malignant diseases and diseases associated with pathological immune responses, such as autoimmune, allergic, inflammatory, and transplantation-related diseases.
NF-κB family molecules are eukaryotic transcription factor complexes critical for the regulation of immune responses, cell growth, and survival (Ghosh S. et al., 1998 Annu Rev Immunol. 16:225-60) which are inducibly activated by almost all TNF/NGF receptor family members. NF-κB molecules are normally sequestered in the cytoplasmic compartment by physical association with a family of cytoplasmic ankyrin-rich inhibitors termed IκB, including IκBα and related proteins (Baldwin A S. Jr., 1996. Annu Rev Immunol. 14:649-83). In response to diverse stimuli, including cytokines, mitogens, and certain viral gene products, IκB is rapidly phosphorylated at Ser32 and Ser36, and is ubiquitinated and subsequently degraded by the 26S proteasome. This allows the liberated NF-κB to translocate to the nucleus and participate in target gene transactivation (Mercurio F. and Manning A. M., 1999. Curr Opin Cell Biol. 11:226-32; Pahl, H. L., 1999. Oncogene 18:6853-66). Recent molecular cloning studies have identified a multi-subunit IκB kinase (IKK) complex that mediates the signal-induced phosphorylation of IκB, the inhibitor of NF-κB. The IKK complex is composed of two catalytic subunits, IKKα and IKKβ, and a regulatory subunit IKKγ (NEMO). The catalytic activity of both IKKα and IKKβ can be activated by a multitude of different NF-κB inducers, including inflammatory cytokines such as tumor necrosis factor (TNF) and interleukin (IL)-1, the T-cell receptor (TCR) and the T-cell costimulatory protein, CD28 (Karin, M. and Ben-Neriah, Y., 2000. Annu Rev Immunol. 18:621-63).
NF-κB-inducing kinase (NIK)/MAP3K-14 (WIPO Pub. No. WO9737016A1 to the present inventors) is critical for activation of NF-κB. For example, overexpression of NIK has been shown to lead to dramatic activation of NF-κB (reviewed in Wallach D. et al., 2002. Arthritis Res. 4 Suppl 3:S189-96), and expression of catalytically-inactive NIK mutants has been shown to lead to effective inhibition of NF-κB activation in response to a variety of known NF-κB activators, such as LMP1, TNF receptor (TNFR)-1, TNFR-2, RANK, human Toll receptor, CD3/CD28, IL-1 receptor (IL-1R), human T-cell lymphotropic virus (HTLV)-1 Tax protein, and lipopolysaccharide (LPS) (Malinin, N. L. et al., 1997. Nature 385:540-4; Sylla, B. S. et al., 1998. Proc Natl Acad Sci USA. 95:10106-11; Darnay, B. G. et al., 1999. J Biol Chem. 274:7724-31; Lin, X. et al., 1999. Immunity 10:271-80; Geleziunas, R. et al., 1998. Mol Cell Biol. 18:5157-65). Targeted disruption of the NIK gene (Yin, L. et al., 2001. Science 291:2162-5), and study of the ‘alymphoplasia’ (aly) mouse strain bearing a natural Gly855Arg missense point mutation in NIK (Shinkura, R. et al., 1999. Nat Genet. 22:74-7) revealed that NIK has an essential role in lymphoid organ development. Both aly/aly and NIK knockout mice manifest systemic absence of lymph nodes and Peyer's patches, disorganized splenic and thymic architectures, and immunodeficiency whose most resilient features are low serum immunoglobulin levels and lack of graft rejection (Shinkura, R. et al., 1999. Nat Genet. 22:74-7). These abnormalities apparently reflect aberrant signaling by a variety of receptors. The developmental deficiencies of the NIK mutant mice resemble those found in mice deficient in the LT-β receptor (LT-βR) suggesting that NIK also participates in signaling by this particular receptor. Impaired B cell proliferative capacity in the aly/aly mice could be shown to correlate to a deficient response of these cells to LPS and CD40 ligand (CD40L; Garceau, N. et al., 2000. J Exp Med. 191:381-6), and presence of excessive amounts of B1 cells in the peritoneal cavity of mice could be ascribed to defects in homing of peritoneal cells to the gut-associated lymphatic tissue (GALT) system as a consequence of deficient chemokine receptor signaling in secondary lymphoid tissue (Fagarasan, S. et al., 2000. J Exp Med. 191:1477-86).
An important and general role for NIK in cytokine receptor signaling has been recently demonstrated in studies performed by Wallach et al. who have shown, using a two-hybrid system, that the IL-2 receptor γ chain, or “common γ chain” which is a signaling component the receptors for IL-2, -4, -7, IL-9, -13, -15 and -21, specifically associates with NIK (PCT/IL 03/00317). Overexpression of the common γ chain was found to potentiate NF-κB activation mediated by NIK, and upon IL-2 or IL-15 stimulation, NIK and signalosome components were found to bind to common γ chain. These results therefore indicate involvement of NIK in signaling via the large variety of cytokine receptors comprising common γ chain as a signaling subunit.
Apart from these and other contributions to the regulation of the development and function of the immune system, NIK is also involved in the regulation of various non-immune functions. The aly/aly (though not the NIK knockout) mice display deficient mammary gland development (Miyawaki, S. et al., 1994. Eur J Immunol. 24:429-34). Moreover, in-vitro studies have implicated NIK in signaling leading to skeletal muscle cell differentiation (Canicio, J. et al., 2001. J Biol Chem. 276:20228-33), and to survival and differentiation of neurons (Foehr, E. D. et al., 2000. J Biol Chem. 275:34021-4).
Consistent with the suggested role of NIK as mediator of NF-κB activation, fibroblasts derived from aly/aly and NIK knock-out mice fail to activate NF-κB in response to LT-βR activation. Moreover, LT-βR upregulation of VCAM-1, which occurs through NF-κB activation, is abnormal in aly/aly mouse embryonic fibroblasts (MEFs; Matsumoto, M. et al., 1999. J Immunol. 163:1584-91). Deficient phosphorylation of IκB has also been noted in the response of aly/aly B-lymphocytes to CD40 ligation. In contrast, in dendritic cells of these mice, CD40-induced phosphorylation of IκB appeared normal (Garceau, N. et al., 2000. J Exp Med. 191:381-6). Aly/aly peritoneal cells are also incapable of responding to the chemokine SLC with increased NF-κB activity (Fagarasan, S. et al., 2000. J Exp Med. 191:1477-86).
Assessment of the pattern of expressed NF-κB species in lymphoid organs of aly/aly mice indicated that, apart from its role in the regulation of NF-κB complex(es) comprised of Rel proteins (A+p50) and IκB, NIK also participates in controlling the expression/activation of other NF-κB species. Most notably, lymphocytes of aly/aly are deficient for p52, an NF-κB species that is specifically formed in mature B lymphocytes through proteolytic processing of an inactive precursor, p100 (NF-κB2), suggesting a deficiency in p100 to p52 conversion (Yamada, T. et al., 2000. J Immunol. 165:804-12). Indeed, NIK has been shown to participate in site-specific phosphorylation of p100. Both directly lead to phosphorylation of IKKα which in turn phosphorylates p100. This phosphorylation serves as a molecular trigger for ubiquitination and active processing of p100 to form p52. This p100 processing activity was found to be ablated by the aly mutation (Xiao, G. et al., 2001. Mol Cell 7:401-9; Senftleben, U. et al., 2001. Science 293:1495-9).
In view of the structural homology of NIK to MAP3Ks, some attempts have been made to explore the involvement of NIK in the ERK, JNK and p38 cascades, the three other main protein kinase cascades involving MAP3Ks (Akiba, H. et al., 1998. J Biol Chem. 273:13353-8). NIK has been shown to be involved in the ERK cascade in PC12 phaeochromocytoma cells (Foehr, E. D. et al., 2000. J Biol Chem. 275:34021-4). Evidence has also been presented that in certain cells NIK may participate in signaling for phosphorylation of Jun, the downstream target of the JNK cascade, independently of this particular cascade (Akiba, H. et al., 1998. J Biol Chem. 273:13353-8; Natoli, G. et al., 1997. J Biol Chem. 272:26079-82). Overall, these findings indicate that NIK indeed serves as a mediator of NF-κB activation, but may also serve other functions, and that it exerts these functions in a cell- and receptor-specific manner.
Similarly to other MAP3Ks, NIK can be activated as a consequence of phosphorylation of the ‘activation loop’ within the NIK molecule. Indeed, mutation of a phosphorylation-site within this loop (Thr559) prevents activation of NF-κB upon NIK overexpression (Lin, X. et al., 1999. Immunity 10:271-80). In addition, the activity of NIK seems to be regulated through the ability of the regions upstream and downstream of its kinase motif to bind to each other (Lin et al. Molec. Cell Biol. (18) 10 5899-5907 1998). The C-terminal region of NIK downstream of its kinase moiety has been shown to be capable of binding directly to IKKα (Regnier, C. H. et al., 1997. Cell 90:373-83) as well as to p100 (Xiao, G. and Sun, S. C., 2000. J Biol Chem. 275:21081-5) and to TRAF2 (Malinin, N. L. et al., 1997. Nature 385:540-4). Such interactions are apparently required for NIK function in NF-κB signaling. The N-terminal region of NIK contains a negative-regulatory domain (NRD), which is composed of a basic motif (BR) and a proline-rich repeat motif (PRR) (Xiao, G. et al., 2001. Mol Cell 7:401-9). Apparently, the N-terminal NRD interacts with the C-terminal region of NIK in cis, thereby inhibiting the binding of NIK to its substrate (IKKα and p100). Ectopically expressed NIK seems to spontaneously form oligomers in which these bindings of the N-terminal to the C-terminal regions in each NIK molecule are apparently disrupted, and display a high level of constitutive activity (Lin, X. et al, 1999. Immunity 10:271-80). The binding of the NIK C-terminal region to the TNF-receptor-associated adaptor protein TRAF2, as well as to other TRAFs, (Malinin, N. L. et al., 1997. Nature 385:540-4; Rothe, M. et al., 1994. Cell 78:681-92; Takeuchi, M. et al., 1996. J Biol Chem. 271:19935-42) most likely participates in the activation of NF-κB by NIK.
Evidence has been presented that NIK, through the binding of its C-terminal region to IKKα, can activate the IKK complex. It has been shown to be capable of phosphorylating Ser176 in the activation loop of IKKα and to thereby activate this molecule (Ling, L. et al, 1998. Proc Natl Acad Sci USA. 95:3792-7). Consistently with such a mode of action, studies regarding the mechanisms accounting for the deficiency in NF-κB activation by LT-βR in aly/aly MEFs have indicated that the NIK mutation ablates activation of the IKK signalosome and the consequent phosphorylation of IκB (Matsushima, A. et al, 2001. J Exp Med. 193:631-6). The ability of NIK to bind p100 directly through its C-terminal region and phosphorylate it suggests that p100 serves as a direct NIK substrate (Xiao, G. and Sun, S. C., 2000. J Biol Chem. 275:21081-5). Nevertheless, a recent study has suggested that NIK mediates p100 phosphorylation in an indirect way, through phosphorylation and thus activation of IKKα that in turn phosphorylates p100 (Senftleben, U. et al, 2001. Science 293:1495-9).
Thus, activation of NF-κB molecules by NIK represents a critical control point for regulation of NF-κB activity.
As described above, disregulated NF-κB activity is associated with the pathogenesis of various major human diseases, such as numerous malignant diseases and diseases associated with pathological immune responses (reviewed in Yamamoto and Gaynor, 2001. J Clin Invest. 107:135-142). For example, activation of the NF-κB pathway is prominently involved in the pathogenesis of chronic inflammatory disease, such as asthma, and rheumatoid arthritis (Tak and Firestein, 2001. J Clin Invest. 107:7-11; Karin, M. and Ben-Neriah, Y., 2000. Annu Rev Immunol. 18:621-63), and inflammatory bowel disease. In addition, altered NF-κB regulation appears to be involved in the pathogenesis of other diseases, such as atherosclerosis (Collins and Cybulsky, 2001. J Clin Invest. 107:255-64; Leonard, W. J. et al., 1995. Immunol Rev. 148:97-114) and Alzheimer's disease (Mattson and Camandola, 2001. J Clin Invest. 107:247-54; Lin, X. et al., 1999. Immunity 10:271-80), in which the inflammatory response is at least partially involved.
Several lines of evidence indicate that NF-κB activation of cytokine genes is an important contributor to the pathogenesis of asthma, which is characterized by the infiltration of inflammatory cells and the deregulation of many cytokines and chemokines in the lung (Ling, L. et al., 1998. Proc Natl Acad Sci USA. 95:3792-7). Cytokines, such as TNF, that activate NF-κB are elevated in the synovial fluid of patients with rheumatoid arthritis and contribute to the chronic inflammatory changes and synovial hyperplasia seen in the joints of these patients (Malinin, N. L. et al., 1997. Nature 385:540-4). The administration of antibodies directed against TNF or a truncated TNF receptor that binds to TNF can markedly improve the symptoms of patients with rheumatoid arthritis.
Increases in the production of proinflammatory cytokines by both lymphocytes and macrophages have also been implicated in the pathogenesis of inflammatory bowel diseases, including Crohn's disease and ulcerative colitis (Matsumoto, M. et al., 1999. J Immunol. 163:1584-91). NF-κB activation is seen in mucosal biopsy specimens from patients with active Crohn's disease and ulcerative colitis. Treatment of patients suffering from inflammatory bowel diseases with steroids decreases NF-κB activity in biopsy specimens and reduces clinical symptoms. These results indicate that stimulation of the NF-κB pathway is involved in the enhanced inflammatory response associated with these diseases.
Atherosclerosis is triggered by numerous insults to the endothelium and smooth muscle of the damaged vessel wall (Matsushima et al., 2001). A large number of growth factors, cytokines, and chemokines released from endothelial cells, smooth muscle, macrophages, and lymphocytes are involved in this chronic inflammatory and fibroproliferative process (Matsushima, A. et al., 2001. J Exp Med. 193:631-6). NF-κB regulation of genes involved in the inflammatory response and in the control of cellular proliferation is widely understood as playing an important role in the initiation and progression of atherosclerosis.
As described above, abnormalities in the regulation of the NF-κB pathway have been shown to be involved in the pathogenesis of Alzheimer's disease. For example, NF-κB immunoreactivity is found predominantly in and around early neuritic plaque types in Alzheimer's disease, whereas mature plaque types show vastly reduced NF-κB activity (Mercurio F. and Manning A. M., 1999. Curr Opin Cell Biol. 11:226-32). Thus, NF-κB activation is in the initiation of neuritic plaques and neuronal apoptosis during the early phases of Alzheimer's disease. These data therefore indicate that activation of the NF-κB pathway plays a role in a number of diseases that have an inflammatory component involved in their pathogenesis.
In addition to a role in the pathogenesis of diseases associated with disregulated immune responses, hyper-activation or constitutive activation of the NF-κB pathway has also been implicated in the pathogenesis of various human cancers. Abnormally high and/or constitutive activation of the NF-κB pathway is frequently seen in a variety of human malignancies including leukemias, lymphomas, and solid tumors (Miyawaki, S. et al., 1994. Eur J Immunol. 24:429-34). These abnormalities result in abnormally and/or constitutively high levels of NF-κB in the nucleus of a variety of tumors including breast, ovarian, prostate, and colon cancers. The majority of these changes are likely due to alterations in regulatory proteins that activate signaling pathways that lead to activation of the NF-κB pathway. However, mutations that inactivate IκB proteins, in addition to amplification and rearrangements of genes encoding NF-κB family members, can result in the enhanced nuclear levels of NF-κB seen in some tumors (Emmerich et al. Blood November 1; 94 (9):3129-34, 1999).
Since, as described above, NIK is critical for activation of NF-κB, one potentially potent strategy for regulating activation of NF-κB, and thereby for treating diseases associated with disregulated NF-κB activity, involves identifying antibodies capable of specifically binding NIK or a specific portion thereof, and of thereby preventing or inhibiting activation of NF-κB by NIK. By virtue of enabling specific detection of NIK or a specific portion thereof, such antibodies would further enable characterization of aspects of normal/pathological biological/biochemical processes/states involving NIK or a specific portion thereof.
Various prior art approaches attempting to use antibodies for specifically binding NIK have been proposed (see Table 2).
One approach has attempted using mouse monoclonal IgG antibodies for specifically binding amino acid residues 700-947 of the carboxy-terminal region of the NIK polypeptide.
Another approach has attempted employing goat polyclonal antibodies for specifically binding the amino-terminal region of the NIK protein.
Yet another approach has attempted utilizing Rabbit polyclonal antibodies for specifically binding amino acid residues 700-947 or 931-947 of the carboxy-terminal region of the NIK polypeptide.
However, all of the aforementioned approaches suffer from significant disadvantages. Prior art antibodies cannot bind NIK, or a specific portion thereof, with optimal affinity, specificity, and/or versatility. For example, prior art NIK-binding antibodies are incapable of specifically binding any portion of the kinase domain of NIK, or of specifically binding numerous portions of the carboxy- and amino-terminal regions, such as portions thereof involved in NIK activity. Hence, since activation of NF-κB involves NIK-mediated phosphorylation events, prior art antibodies are not suitable for preventing or inhibiting the kinase activity of NIK. Prior art polyclonal antibody preparations are further hampered by their having been raised against suboptimally large NIK segments, and thereby by including antibodies specific for a suboptimally broad range of epitopes (for example, refer to Table 2, below). Furthermore, prior art non-mouse derived/anti-amino-terminal region antibodies cannot be specifically bound by anti-mouse antibody ligands which constitute the most widely employed type of secondary labeling reagents used in antibody-based detection assays. As such, prior art anti-amino terminal region antibodies are suboptimal for detecting the amino-terminal region of NIK.
Thus, all prior art approaches have failed to provide antibodies capable of binding NIK, or a specific portion thereof, with an affinity, specificity and/or versatility enabling optimal regulation of NIK activity, and hence of NF-κB activity, and/or enabling optimal detection of NIK.
There is thus a widely recognized need for, and it would be highly advantageous to have, antibodies devoid of the above limitations.